High-frequency genetic transformation of
Phalaenopsis amabilis
orchid using tomato extract-enriched medium for the pre-culture
of protocorms
By E. SEMIARTI
1, 2, A. INDRIANTO
1, A. PURWANTORO
3, I. N. A. MARTIWI
1,
Y. M. L. FERONIASANTI
1, F. NADIFAH
1, I. S. MERCURIANA
2, R. DWIYANI
2,
H. IWAKAWA
4, Y. YOSHIOKA
5, Y. MACHIDA
5and C. MACHIDA
4, 6*
1
Faculty of Biology, Gadjah Mada University, Jl. Teknika Selatan, Sekip Utara, Yogyakarta 55281,
Indonesia
2
Center Study of Biotechnology, Gadjah Mada University, Barek, Yogyakarta 55281, Indonesia
3
Faculty of Agriculture, Gadjah Mada University, Bulaksumur, Yogyakarta 55281, Indonesia
4Plant Biology Research Center, Chubu University, 1200 Matsumoto-cho, Kasugai, Aichi 487-8501,
Japan
5
Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho,
Chikusa-ku, Nagoya 464-8602, Japan
6
College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai,
Aichi 487-8501, Japan
(e-mail: [email protected])
(Accepted 1 February 2010)
SUMMARY
The development of an efficient methodology for the genetic transformation of orchids is needed in order to support the genetic engineering of orchids. It is therefore important to identify those factors affecting the transformation process. Previously, we reported a convenient method for the transformation of Phalaenopsis amabilis using Agrobacterium tumefaciens, in which intact protocorms were used. We also found that embryos cultured on a medium containing tomato extract grew more rapidly than those cultured on a medium with coconut water. When we used protocorms grown on a medium containing tomato extract, we obtained regenerated shoots that had been transformed with a kanamycin resistance gene at relatively high frequencies (7 – 17%). These results suggest that the rate of growth of pre-cultured protocorms may be important for the successful regeneration of transformed shoots. We also obtained regenerated shoots that had been transformed with the green fluorescent protein (GFP) gene at a high frequency (10 – 14%). Both the presence and expression of these transgenes were confirmed in transformed plants by molecular analyses and by the detection of green fluorescence following excitation with blue light.
I
t is important and of practical value for the orchid industry to generate novel traits such as improved floral characters to satisfy consumer demand and appreciation for aesthetics and novelty. A common trend in current orchid biotechnology is the application of molecular techniques for orchid improvement, which allows the introduction of desirable traits by introducing specific genes. The core component of the molecular breeding of orchids is the need to create efficient and reproducible gene transformation systems. A reproducible methodology for the genetic transformation of orchids, and better recognition of the factors affecting the transformation process, are needed in order to support this objective.Previous studies have reported orchid transformation either directly through the delivery of marker genes such as those encoding Escherichia coli -glucuronidase (GUS) and Aequorea victoriagreen fluorescent protein (GFP) into plant cells by particle bombardment (Anzai et al. 1996), or indirectly through the use of
Agrobacterium tumefaciens (Belarmino and Mii, 2000; Chia et al., 1994; Mishiba et al., 2005; Chan et al., 2005; Sjahril et al., 2006; Sjahril and Mii, 2006). Recently, we have developed a convenient method for genetic modification of Phalaenopsis amabilis orchid using A. tumefaciens (Semiarti et al., 2007) in which intact protocorms (young orchid seedlings) were used for transformation. This method is also simple, reproducible, and applicable in other species. However, the transformation efficiency was ≤2%, and further studies were needed to improve this.
completely by using these anti-oxidants, while the virulence of Agrobacteriumremained unaffected. These treatments enabled the recovery of stable transgenic grapevine plants resistant to hygromycin.
In the present study, in order to improve the frequency of Agrobacterium-mediated transformation of P. amabilis, we pre-cultured the protocorms on medium containing an extract from fully-ripe tomato fruit and investigated the effect of this pre-culturing treatment on improving the efficiency of regeneration of transformed shoots.
MATERIALS AND METHODS
Plant material, growth conditions, and culture medium Adult plants of Phalaenopsis amabilis (L.) Blume from Java were obtained from Royal Orchids (Prigen, East Java, Indonesia). Seeds were derived from cross-pollinated plants that had been sown on a modified, new Phalaenopsis (NP) medium (Islam et al., 1998) and maintained under continuous white light. Adult plants were maintained in a glasshouse at room temperature. Seeds were sown on modified NP medium with various concentrations of coconut water (50 – 150 ml l–1
) and/or tomato extract (50 – 200 mg l–1
) and grown for 3 weeks to
produce protocorms, which were used for
transformation. Coconut (Cocos nuciferafrom Java) and the tomato (Lycopersicon esculentum) cultivar ‘Arthaloka’ from West Java were obtained from local markets. Tomato fruit extract was prepared by cutting tomatoes into 1 cm3
cubes, homogenising them, and filtering the homogenate through a steel mesh with a 150-µm pore size. The nutrient compositions of the coconut water sample and the tomato extract were analysed by high performance liquid chromatography at the Food and Product Technology Laboratory of the Faculty of Agricultural Technology of Gadjah Mada University.
Developmental stages of P. amabilis
To determine the growth rates of orchid embryos and protocorms, the sizes, colours, and shapes of the embryos or protocorms were evaluated as described by Dressler (1981). At Stage 0, each intact seed (270 – 400 µm-long) with its embryo (100 – 200 µm-long) is coated by a layer of net-like cells, the testa. At Stage 1, the testa spreads apart and the embryo swells into an ovoid-shaped mass of cells. At Stage 2, the seed coat cracks and the mass of cells grows outside the coat (0.5 – 1.0 mm-long). At Stage 3, the mass of cells elongates gradually into a cone-shaped body (1.0 – 1.4 mm-long). At Stage 4 (the protocorm), root hairs emerge from the basal portion of the cone-shaped body, which turns green. At Stage 5, the photosynthetic protocorm forms a leafy shoot at its apex and forms new root hairs. After Stage 5, seed germination is complete, two leaves gradually emerge and roots form.
Plasmid vector and bacterial strain
Using the binary plasmid vector pBI121 (Clontech Laboratories Inc., Otsu, Japan), containing a kanamycin resistance gene and the 35S CaMV promoter with the 3'nosterminator, a PCR-amplified fragment containing the entire coding region of the GFPgene was used to
generate a plasmid that we designated pBI121-p35S::GFP. This construct was introduced into the disarmed, octopine-type A. tumefaciensstrain LBA4404 (Hoekema et al., 1983).
Nucleic acid isolation and purification
Nucleic acids (genomic DNA, total RNA, mRNA, and cDNA) were prepared according to Semiarti et al. (2007).
Transformation and transformant regeneration
Transformants were obtained and regenerated using the methods described by Semiarti et al. (2007). Genomic DNA from putative 35S::GFP transformants was analysed by PCR using the following selective forward (F) and reverse (R) primers to detect both the kanamycin resistance gene (neomycin phosphotransferase II; NPTII) and the GFP gene: NPTIIF1 CCTGCCCATTCGACCACCAA-3’) and NPTIIR1
(5’-AGCCCCTGATGCTCTTCGTC-3’) for the NPTII
gene; and GFPF1 (5’-ATGGTGAGCAAGGGCGAG GA-3’) and GFPR1 (5’-GTCCATGCCGTGAGTG ATCC-3’) for the GFPgene. PCR was performed with 30 cycles of 94°C for 1 min, 60°C for 30 s, and 72°C for 90 s. As an internal control, genomic DNA was amplified using primers for the ACTIN gene, as described by Semiarti et al. (2007). To detect GFPgene expression in the transformants, seedlings or plant tissues were excited with blue light (495 nm) using a Nikon Diaphot 300 microscope (Nikon Corp., Tokyo, Japan) equipped with a B2 filter, which distinguished the red autofluorescence of chlorophyll from the fluorescence of GFP. The images were captured using a Nikon Cool Pix 5000 digital camera system, with adaptor for microscopy (Nikon Corp.).
DNA analysis by Southern hybridisation
Genomic DNA from 9-month-old leaves of five independent transgenic lines of P. amabilisthat expressed GFP fluorescence was digested using the restriction enzymes EcoRI and Hind III. These plants also yielded the predicted 360-bp PCR product using a primer pair designed for the GFP coding region. The digested genomic DNA fragments were transferred to a nylon membrane (Amersham Hybond-N+
; GE Healthcare, Cambridge, UK) and hybridised with a digoxigenin-labelled probe for the GFP gene derived from the plasmid pBI121-GFP (12.6 kbp) using the DIG DNA Labeling Kit (Roche Diagnostics, Tokyo, Japan). The hybridised DNA fragments were visualised using the DIG Luminescent Detection Kit (Roche Diagnostics) according to the manufacturer’s instructions.
RESULTS AND DISCUSSION
Effect of tomato extract on the formation of shoots from protocorms of P. amabilis
We tested coconut water and tomato extract as potential supplements to accelerate the growth of Phalaenopsis embryos, especially at the early developmental Stages, using embryos grown on NP medium with or without either supplement.
I. S. MERCURIANA, R. DWIYANI, H. IWAKAWA, Y. YOSHIOKA, Y. MACHIDAand C. MACHIDA
TABLEI
Growth stages of P. amabilisseeds cultured on NP medium supplemented with different concentrations of tomato extract at 21 days after sowing
Protocorm Concentration Total no. Swollen embryo Green with shoot of tomato embryos Experiment Embryo (protocorm) protocorm apical meristem extract (mg l–1) examined No. Stage 0 (%) Stage 1 (%) Stage 2 (%) Stage 3 (%) Stage 4 (%) Stage 5 (%)
0 806 1 23.9 12.2 3.6 20.8 39.1 0.5
2 18.7 5.4 7.4 30.5 36.9 1.0
3 24.4 2.2 4.9 40.1 28.3 0.0
50 1,148 1 22.2 3.6 4.3 40.2 29.3 0.4
2 27.5 6.0 8.8 28.4 28.4 0.9
3 28.0 6.9 4.0 28.7 31.5 0.9
100 1,577 1 24.9 4.7 5.5 13.0 51.1 0.8
2 31.3 1.9 6.1 9.0 51.2 0.5
3 36.0 4.1 2.0 15.8 42.1 0.0
150 1,417 1 46.0 4.8 3.7 9.5 36.1 0.0
2 26.2 1.8 5.6 14.6 51.8 0.0
3 35.5 2.9 4.4 15.0 42.3 0.0
200 1,583 1 49.6 3.1 2.8 9.7 34.7 0.0
2 62.6 7.8 3.6 6.0 20.1 0.0
3 52.5 5.7 1.1 17.0 23.7 0.0
FIG. 1
Main Panel A, growth of protocorms of P. amabilison various culture media 3 weeks after sowing. Sub-Panel a, NP medium; sub-Panel b, NP medium supplemented with 150 ml l–1coconut water (CW); sub-Panel c, NP medium supplemented with 100 mg l–1tomato extract (TE); and sub-Panel d, NP
medium supplemented with 150 ml l–1CW and 100 mg l–1TE. Main Panel B, development of shoots from protocorms of P. amabilisthat had been
cultured on NP medium supplemented with coconut water (CW) and/or tomato extract (TE) for 3 weeks. Protocorms were selected from NP medium containing 200 mg l–1kanamycin after Agrobacterium-mediated transformation with pBI121 after 5 weeks. Sub-Panel a, unregenerated protocorms
on medium containing 200 mg l–1kanamycin (Km); sub-Panels b–d, kanamycin-resistant seedlings produced from protocorms that had been
transformed with pBI121 containing the kanamycin resistance gene (NPTII). Main Panel C, PCR detection of the kanamycin resistance gene (NPTII) in putative transgenic orchid plants harbouring pBI121. Fragments from a StyI digest of phage DNA were used as size markers (M). No amplified DNA fragments from the kanamycin resistance gene were seen in DNA from untransformed P. amabilisorchid plants (lane 1). The specific 105-bp PCR fragment of the NPTIIgene was amplified from DNA of putative transgenic orchid seedlings from sub-Panels b (lanes 2 –7), c (lanes 8 – 12),
tomato extract based on the number of growing embryos and protocorms found at each Stage (Table I). The number of seeds developing to Stage 4 was increased at higher concentrations of tomato extract in the NP medium, achieving an optimal number at 100 – 150 mg l–1
tomato extract. Therefore, 100 mg l–1
tomato extract was used in the following experiments.
We also analysed growth rates on NP medium with or without coconut water and tomato extract (Figure 1A). The fastest rate of embryo development was observed on NP medium supplemented with both coconut water and tomato extract. Protocorms cultured on NP medium containing tomato extract alone appeared to change from yellow to green more rapidly than those cultured on NP medium containing coconut water alone. Tomato extract thus appeared to affect the growth rate at all Stages of embryo development, including the formation of the shoot apical meristem prior to the emergence of the leaf primordia. The tomato extract contained carotene, vitamin C, and other anti-oxidants which were not detected in coconut water (Table II). These components could affect growth of the embryo.
Oladiran and Iwu (1992) showed that fully-ripe tomato fruit contained basic nutrients and essential vitamins, as well as trace elements. Among these, carotenoids with cyclic end-groups were essential components of all photosynthetic membranes and played several roles, including protection against photo-oxidation (Cunningham et al., 1996). These are potential candidates for the growth-promoting compounds in the tomato extract, as it was rich in carotenoids. We therefore tested a single carotenoid, lycopene, for its possible effects on growth promotion, but found no significant effect at concentrations typically found in tomato extracts [≤ 0.1% (w/w)], while high concentrations of lycopene inhibited seed growth (data not shown). Further studies on other components found in tomato extract are needed to determine whether any single compound has an effect, or several compounds have a synergistic effect, on the growth and development of P. amabilisseed.
Effect of pre-culture of protocorms on NP medium containing tomato extract on the transformation frequency of P. amabilis
Protocorms were pre-cultured on NP medium supplemented with coconut water and/or tomato extract, prior to transformation, to determine the effects of pre-culture supplementation on the frequency of transformation (Table III). The transformation efficiency was determined based on the percentage of protocorms that produced shoots on the selective medium out of the total number of protocorms examined. The transformation frequency of regenerated shoots was increased from 1.2% on NP medium with coconut water alone to 13.2% on NP medium containing 100 mg l–1
tomato extract alone, and to between 6.8 – 16.6% on NP medium containing both coconut water and tomato extract (Table III; Figure 1B, Panel D). These results were higher than the frequency of transformed regenerated shoots on medium containing coconut water alone (1.2%; Table III), confirming the observations made by Semiarti et al. (2007).
In the case of transformation with pBI121-p35S::GFP, transformed regenerated shoots were produced at frequencies of 9.8 – 13.5% following pre-culture on NP medium supplemented with both coconut water and tomato extract (Table III). Overall, the transformation frequencies of protocorms pre-cultured on NP medium supplemented with tomato extract alone, or with both coconut water and tomato extract were higher than that of protocorms pre-cultured on NP medium supplemented with coconut water alone, suggesting that the growth rate of protocorms was related to the pre-culture conditions which are therefore important for the regeneration of transformed shoots.
Several studies have examined the use of rich sources of nutrients, vitamins, and phytohormones, including coconut water, carrot, maize, or potato extracts, as possible supplements for stimulating the germination of various orchid species (Arditti and Ernst, 1993; Raghavan, 1997; Islam et al., 2003; Mishiba et al., 2005; Chansean and Ichihashi, 2007). More studies on other sources of nutrients may be required to establish an optimum method for transformation.
Molecular analysis of putative transformants
First, we examined the genomic DNA from P. amabilis plantlets regenerated on agar plates containing 200 mg l–1
kanamycin for the presence and expression of the kanamycin resistance gene (NPTII) using PCR. The predicted 105-bp fragment was amplified from all putative transformants in each treatment (Figure 1C).
TABLEIII
Transformation frequency of P. amabilisprotocorms following 3 weeks of pre-culture on NP medium supplemented with tomato extract and/or
coconut water
No. protocorms Total no. producing Coconut Tomato protocorms shoots Plasmid water extract examined (% of total) None + + 1,557 0 (0%) pBI121 (vector) + – 1,200 14 (1.2%) pBI121 (vector) – + 1,200 159 (13.2%) pBI121 (vector) + + 1,557† 260 (16.6%)
+ + 1,500† 102 (6.8%)
pBI121-p35S::GFP + + 1,557‡ 210 (13.5%)
+ + 1,500‡ 147 (9.8%) †, ‡Data are from two independent transformation experiments in each
case. TABLEII
Components of coconut water and tomato extract used in this study
Coconut water Tomato extract
*DPPH, 1,1-diphenyl-2-picrylhydrazyl, an ingredient that prevents oxidation.
The samples used were coconut water from Cocos nuciferafrom Java and an extract from the tomato cultivar ‘Arthaloka’ (Lycopersicon esculentum) from West Java. All values are percentages (w/w). All samples contained 91 – 95% (w/w) H2O. Values are based on the average
I. S. MERCURIANA, R. DWIYANI, H. IWAKAWA, Y. YOSHIOKA, Y. MACHIDAand C. MACHIDA
The plantlets that regenerated after transformation with the plasmid pBI121-p35S::GFP were examined for the presence of the GFPgene by PCR amplification of the 360-bp fragment from the GFP coding region (Figure 2B). Of the 210 plantlets examined, 191 were positive for the GFPgene fragment.
To confirm the presence of the GFP gene, and to assess the gene copy number in plants that also showed kanamycin resistance, we performed Southern hybridisations. Hybridisation using an anti-sense probe for the 3' end of the GFPgene (Figure 2A) showed two-to-four copies of the GFPgene in each transgenic line (Figure 2C). Since the genomic DNA of each putative transgenic plant showed uniquely-sized bands hybridising to the GFPanti-sense fragment, this T-DNA fragment was confirmed to be inserted into the genome
at different independent sites, and in multiple copies in each putative transgenic plant line.
For further analysis, we purified total poly(A)+
RNA from individual leaves of an untransformed wild-type plant, a plantlet transformed with pBI121, and three lines transformed with pBI121-p35S::GFP. We quantified the relative levels of GFPgene transcripts (mRNA) using RT-PCR with the primers specific for GFP. PCR products were detected in all three lines of plantlets transformed with pBI121-p35S::GFP, but not in the untransformed plantlet or the plantlet transformed with pBI121 alone (Figure 2D). Thus, transcripts of the GFP gene had accumulated in the leaves of the transformants, confirming expression of the GFPtransgene in these plants. Plantlets transformed with pBI121-p35S::GFP showed green fluorescence after excitation with blue light (Figure 2E),
FIG. 2
Analysis of putative GFPtransformants of P. amabilis. Panel A, schematic representation of the T-DNA region of the binary plasmid vector pBI121-p35S::GFP. The binary plasmid pBI121-p35S::GFP contained the 720-bp GFPgene, which encodes the jellyfish green fluorescent protein under the control of the 35S promoter (P35S) of cauliflower mosaic virus (CaMV). RB, right border; LB, left border; Pnos, promoter of the nopaline synthase gene; Tnos, polyadenylation site of the nopaline synthase gene;NPTII, neomycin phosphotransferase gene; P35S, 35S promoter of CaMV. A 360-bp fragment from the 3’ end of the GFPgene was used as a probe during Southern hybridisation. Panel B, PCR detection of the GFPtransgene in putative transgenic orchids. Fragments from a StyI digest of phage DNA were used as size markers (M). The specific 360-bp DNA fragment of the GFPgene was amplified from the plasmid pBI121-p35S::GFP (lane 1), no fragment was amplified from DNA of an untransformed P. amabilis
plant (lane 2), or a plant transformed with the empty vector pBI121 (lane 3). The 360-bp GFPPCR fragment was seen in genomic DNA from three plants independently transformed with pBI121-p35S::GFP (lanes 4 – 6). Panel C, Southern blot analysis. Fragments from a StyI digest of phage DNA were used as size markers (kbp) on the left-side. Lane GFP, the GFP probe itself. Lane NT, genomic DNA from an untransformed plant was digested with EcoRI. Lane pairs marked T#1, T#2, T#3, and T#4 show genomic DNA (5 µg) from putative transformed plants digested with EcoRI (E) or Hind III (H). Fragments were fractionated in a 1% (w/v) agarose gel, then blotted and hybridised with a digoxigenin (DIG)-labelled GFPgene probe. Panel D, expression of the GFPgene in three putative transgenic P. amabilisplants. RT-PCR analysis of transcripts of the GFPgene in an untransformed plant (NT), in a plant transformed with the empty vector pBI121 (V), and in three independent pBI121-p35S::GFP-transformed plantlets (1 – 3). The number of PCR cycles is indicated on the right of each sub-Panel. Amplified DNA fragments were separated by electrophoresis in an agarose gel and visualised with ethidium bromide. As a control, the same samples were amplified with primers specific for ACTINgene transcripts. (see Materials and Methods for details on RT-PCR). Panels E and F, detection of GFPgene expression in a putative 35S::GFP transformed
whereas untransformed plantlets did not (Figure 2F). Taken together, the molecular analyses of the transformants strongly suggests that supplementation using tomato extract during pre-culture in NP medium improved the transformation efficiency of P. amabilisseveral-fold.
We thank Dr. Saeko Kitakura, Dr. Yoshihisa Ueno, and Mr. Masaya Ikezaki of Nagoya University, Japan, for gifts of the pBI121-p35S::GFP plasmid and for useful discussions. We also thank Dr. André Schuiteman, National Herbarium Netherlands, Leiden University, The Netherlands, and Dr. Shogo Matsumoto, Nagoya
University, for helpful discussions and critical reading of the manuscript. We thank Dr. Hirokazu Tsukaya, University of Tokyo, Japan, for helpful discussions. E.S., A.I. and A.P. were supported by a grant from the Ministry of Research and Technology of the Republic of Indonesia on Project RUT IX (No. 14.15/SK/RUT/2004). This work was also supported, in part, by Grants-in-Aid for Scientific Research on Priority Areas (No. 19060003) to Y.M. and C.M. from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and by an Academic Frontier Project for Private Universities matching fund subsidy from MEXT 2005-2009.
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